U.S. patent application number 12/520715 was filed with the patent office on 2010-06-10 for treatment of macular degeneration.
Invention is credited to Frances Margaret Richards, Alan Geoffrey Roach.
Application Number | 20100143346 12/520715 |
Document ID | / |
Family ID | 37758999 |
Filed Date | 2010-06-10 |
United States Patent
Application |
20100143346 |
Kind Code |
A1 |
Richards; Frances Margaret ;
et al. |
June 10, 2010 |
Treatment of Macular Degeneration
Abstract
An agent having progesterone antagonist properties may be used
to treat eye conditions associated with pathological blood vessel
formation, for example age-related macular degeneration, choroidal
neovascularisation, retinal neovascularisation or corneal
neovascularisation. The agent may be mifepristone.
Inventors: |
Richards; Frances Margaret;
(Oxfordshire, GB) ; Roach; Alan Geoffrey;
(Oxfordshire, GB) |
Correspondence
Address: |
SALIWANCHIK LLOYD & SALIWANCHIK;A PROFESSIONAL ASSOCIATION
PO Box 142950
GAINESVILLE
FL
32614
US
|
Family ID: |
37758999 |
Appl. No.: |
12/520715 |
Filed: |
December 21, 2007 |
PCT Filed: |
December 21, 2007 |
PCT NO: |
PCT/GB07/04947 |
371 Date: |
February 19, 2010 |
Current U.S.
Class: |
424/133.1 ;
514/170; 514/171; 514/179; 514/2.4; 514/44R; 514/8.1 |
Current CPC
Class: |
A61K 31/567 20130101;
A61K 31/575 20130101; A61P 9/00 20180101; A61P 33/02 20180101; A61P
27/14 20180101; A61P 27/10 20180101; A61P 31/04 20180101; A61K
45/06 20130101; A61P 43/00 20180101; A61P 27/02 20180101; A61P
31/22 20180101; A61K 31/00 20130101; A61P 27/06 20180101; A61K
31/56 20130101; A61K 31/56 20130101; A61K 2300/00 20130101; A61K
31/575 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/133.1 ;
514/44.R; 514/170; 514/171; 514/179; 514/11 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61P 27/02 20060101 A61P027/02; A61P 27/06 20060101
A61P027/06; A61P 27/10 20060101 A61P027/10; A61P 33/02 20060101
A61P033/02; A61P 27/14 20060101 A61P027/14; A61P 31/04 20060101
A61P031/04; A61P 31/22 20060101 A61P031/22; A61K 31/7088 20060101
A61K031/7088; A61K 31/575 20060101 A61K031/575; A61K 31/57 20060101
A61K031/57; A61K 38/12 20060101 A61K038/12 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 22, 2006 |
GB |
0625844.6 |
Claims
1. A method for treating a condition associated with blood vessel
formation wherein said method comprises administering to a subject
in need of such treatment an agent having progesterone antagonist
properties.
2. The method, according to claim 1, wherein the condition is an
ocular condition.
3. The method, according to claim 2, for the treatment for an
ocular condition in which neovascularisation is involved.
4. The method, according to claim 1, wherein the condition is
age-related macular degeneration, choroidal neovascularisation,
retinal neovascularisation, or corneal neovascularisation.
5-7. (canceled)
8. The method, according to claim 1, wherein the condition is
selected from ocular histoplasmosis syndrome, pathologic myopia,
angioid streaks, idiopathic disorders, choroiditis, choroidal
rupture, overlying choroid nevi, Best's disease, Stargardt's
disease, Vogt-Koyanagi-Harada syndrome, toxoplasmosis, sickle cell
disease, diabetic retinopathy and other proliferative retinopathy,
retinopathy of prematurity, neovascular glaucoma, sarcoidosis,
syphilis, pseudoxanthoma elasticum, vein or artery occlusion,
carotid obstructive disease, chronic uveitis/vitritis,
mycobacterial infection, Lyme's disease, Eale's disease, systemic
lupus erythematosus, Behcet's disease, infections causing
retinitis, optic pits, par planitis, chronic retinal detachment,
hyperviscosity syndromes, capillary haemangioma including von
Hippel-Lindau disease, trauma, post-laser complication, epidemic
keratoconjunctivitis, Vitamin A deficiency, contact lens overwear,
atopic keratitis, superior limbic keratitis, pterygium keratitis
sicca, sjogrens, acne rosacea, phylectenulosis, syphilis,
Mycobacteria infections, lipid degeneration, chemical burns,
bacterial ulcers, fungal ulcers, Herpes simplex infections, Herpes
zoster infections, protozoan infections, Kaposi sarcoma, Mooren
ulcer, Terrien's marginal degeneration, marginal keratolysis,
rheumatoid arthritis, systemic lupus, polyarteritis, trauma,
Wegeners sarcoidosis, Scleritis, Steven's Johnson disease,
pemphigoid radial keratotomy, and corneal graft rejection.
9. The method, according to claim 1, wherein the agent is a
progesterone receptor antagonist.
10. The method, according to claim 1, wherein the agent has a
progesterone receptor binding activity of 0.01 nM to 10 .mu.M.
11. The method, according to claim 1, wherein the agent is
mifepristone, onapristone or asoprisnil.
12. The method, according to claim 1, wherein the agent is a
metabolite of mifepristone selected from RU42698, RU 42848, RU42633
and the demethyl and didemethyl derivatives of RU42698.
13. The method, according to claim 1, wherein the medicament is
administered orally or topically.
14. The method, according to claim 13, wherein the medicament is
administered topically to the eye.
15. The method, according to claim 14, wherein the medicament is an
eye drop.
16. The method, according to claim 1, wherein the medicament
comprises a slow release drug delivery system.
17. The method, according to claim 1, wherein the subject of
treatment is also given, topically, systematically or directly into
the eye via injection or implant, another drug selected from
macugen, Lucentis, avastin and other VEGF inhibitors, VEGF receptor
tyrosine kinase inhibitors, protein kinase C inhibitors, inhibitors
of other angiogenic proteins, recombinant angiostatic factors,
somatostatin analogues, corticosteroids, statins (inhibitors of
HMG-CoA reductase), squalamine lactate, thiamine and its analogues,
angiotensin receptor blockers and rapamycin.
18. A method for treating wet AMD wherein said method comprises
administering, to a subject in need of such treatment, mifepristone
(RU486), or a metabolite or pharmaceutically acceptable derivative
thereof.
19. The method of claim 18 wherein the mifepristone (RU486)
metabolite is selected from RU42698, RU42848, RU42633 and the
demethyl and didemethyl derivatives of RU42698.
20. A pharmaceutical composition for topical administration to the
eye comprising mifepristone (RU486), or a metabolite or
pharmaceutically acceptable derivative thereof.
21. The pharmaceutical composition of claim 20 wherein the
mifepristone (RU486) metabolite is selected from RU42698, RU42848,
RU42633 and the demethyl and didemethyl derivatives of RU42698.
22-29. (canceled)
30. The method of claim 18, wherein the subject treated is also
given, topically, systematically or directly into the eye via
injection or implant, another drug selected from macugen, Lucentis,
avastin and other VEGF inhibitors, VEGF receptor tyrosine kinase
inhibitors, protein kinase C inhibitors, inhibitors of other
angiogenic proteins, recombinant angiostatic factors, somatostatin
analogues, corticosteroids, statins (inhibitors of HMG-CoA
reductase), squalamine lactate, thiamine and its analogues,
angiotensin receptor blockers and rapamycin.
Description
FIELD OF THE INVENTION
[0001] This invention relates to the treatment and prophylactic
prevention of conditions associated with undesired blood vessel
formation and in particular ocular conditions characterised by the
presence of unwanted neovascularisation, such as neovascular
age-related macular degeneration and diabetic retinopathy.
BACKGROUND TO THE INVENTION
[0002] Retinal and choroidal neovascularization (CNV) can lead to
hemorrhage and fibrosis, with resulting visual loss in a number of
conditions of the eye, including, for example, age-related macular
degeneration, ocular histoplasmosis syndrome, pathologic myopia,
angioid streaks, idiopathic disorders, choroiditis, choroidal
rupture, overlying choroid nevi, Best's disease, Stargardt's
disease, Vogt-Koyanagi-Harada syndrome, toxoplasmosis, certain
inflammatory diseases, sickle cell disease, diabetic retinopathy
and other proliferative retinopathy, retinopathy of prematurity,
neovascular glaucoma, sarcoidosis, syphilis, pseudoxanthoma
elasticum, vein or artery occlusion, carotid obstructive disease,
chronic uveitis/vitritis, mycobacterial infection, Lyme's disease,
Eale's disease, systemic lupus erythomatosus, Behcet's disease,
infections causing retinitis, optic pits, par planitis, chronic
retinal detachment, hyperviscosity syndromes, capillary haemangioma
including von Hippel-Lindau disease, trauma and post-laser
complication.
[0003] In addition, pathological neovascularisation can occur in
the cornea. Examples of corneal neovascularization include,
epidemic keratoconjunctivitis, Vitamin A deficiency, contact lens
overwear, atopic keratitis, superior limbic keratitis, pterygium
keratitis sicca, sjogrens, acne rosacea, phylectenulosis, syphilis,
Mycobacteria infections, lipid degeneration, chemical burns,
bacterial ulcers, fungal ulcers, Herpes simplex infections, Herpes
zoster infections, protozoan infections, Kaposi sarcoma, Mooren
ulcer, Terrien's marginal degeneration, mariginal keratolysis,
rheumatoid arthritis, systemic lupus, polyarteritis, trauma,
Wegeners sarcoidosis, Scleritis, Steven's Johnson disease,
pemphigoid radial keratotomy, and corneal graft rejection.
[0004] One of the disorders, namely, age-related macular
degeneration (AMD), is the leading cause of severe vision loss in
people aged 65 and above (Leibowitz, Krueger et al. 1980; Klein and
Klein 1982; Bressler, Bressler et al. 1988). Patients with early
AMD have drusen and altered retinal pigmentation, with only subtle
abnormalities in visual function. Patients with late stage AMD have
severe vision loss due to either geographic atrophy ("dry" AMD) or
CNV ("wet" AMD). In CNV, abnormal blood vessels originate from the
choriocapillaris beneath the retina and grow under the retinal
pigment epithelium (RPE) and retina. These new vessels have a
tendency to leak, resulting in distortion of the macula and central
vision loss.
[0005] Vascular Endothelial growth factor (VEGF) plays a key role
in angiogenesis by stimulation of proliferation of endothelial
cells and by permeabilisation of vessels. Other pro-angiogenic
factors are also involved, including fibroblast growth factor,
platelet derived growth factor, insulin-like growth factor,
transforming growth factors alpha and beta, angiopoietin-1 and -2.
There are also anti-angiogenic factors that play a role in
controlling angiogenesis, including pigment epithelial derived
growth factor (PEDF), thrombospondin, angiostatin, and endostatin.
An imbalance in the ratio of angiogenic and anti-angiogenic
factors, including increased VEGF and decreased PEDF, is thought to
contribute to CNV. Induction of VEGF through the hypoxia-response
element in the VEGF promoter is required for development of CNV in
a mouse model (Muranaka, Yanagi et al. 2005; Vinores, Xiao et al.
2006) and it is thought that in AMD outer retina hypoxia may
stimulate VEGF production to drive CNV formation (Schlingemann
2004).
[0006] The potential for anti-VEGF therapy to influence CNV (wet
AMD) has been recognised with the development of several drugs
designed to inhibit VEGF. The anti-VEGF aptamer Pegaptinib
(Macugen) and the anti-VEGF antibodies ranibizumab (Lucentis) and
bevacizumab (Avastin) have shown promise in clinical trials but
these drugs require regular dosing by intravitreal injection, which
is an invasive technique with risks of trauma and endophthalmitis
(Yeoh, Sims et al. 2006). Thus, there is a market for
anti-angiogenic agents that can be administered in a less invasive
manner, ideally by topical administration, or perhaps using a slow
release delivery system.
[0007] Progesterone receptor mRNA has been detected in many
different ocular tissues (Suzuki, Kinoshita et al. 2001;
Fuchsjager-Mayrl, Nepp et al. 2002), including rabbit
retina/choroid (Wickham, Gao et al. 2000) and chick retina
(Lippman, Wiggert et al. 1974) but the cell types in which the
receptor is expressed has not been elucidated and the role of these
receptors in the eye is not clear. The progesterone receptor (PR)
was also demonstrated to function in intact chick retina (Li, Hayes
et al. 1997). Progesterone may have a role in function of the
neuroretina since it is present in the rat retina (Lanthier and
Patwardhan 1988) and it enhances survival of rat retinal ganglion
cells in culture (Lindsey and Weinreb 1994). Also, in isolated
bovine retinal pigment epithelial (RPE) microsomes progesterone
inhibits acyl CoA:retinol acyltransferase (ARAT) (Ross 1982;
Kaschula, Jin et al. 2006), an enzyme which may be a component of
the visual cycle.
[0008] The most commonly known progesterone receptor antagonist is
RU486, i.e.
17beta-hydroxy-11-beta-(4-dimethylaminophenyl)-17alpha-(prop-1-ynyl)-
-estra-4,9-dien-3-one, also known as mifepristone, RU38486 and
C-1073. RU486 competes with endogenous progesterone by high
affinity competitive receptor binding. It also acts as a high
affinity competitive inhibitor for the glucocorticoid receptor,
binding with two to three times the affinity of dexamethasone
(Moguilewsky and Philibert 1984; Gagne, Pons et al. 1985). RU486 is
used clinically as an abortifacient and has been investigated
extensively for use as a contraceptive (Baulieu and Ulmann 1986;
Sitruk-Ware and Spitz 2003). It has also been tested in clinical
trials for psychotic depression (Simpson, El Sheshai et al. 2005;
Flores, Kenna et al. 2006), Alzheimer's disease (DeBattista and
Belanoff 2005), schizophrenia (Gallagher, Watson et al. 2005),
uterine myomas (Eisinger, Bonfiglio et al. 2005), endometriosis
(Kettel, Murphy et al. 1996), leiomyoma (Fiscella, Eisinger et al.
2006), meningioma (Spitz, Grunberg et al. 2005) and
hormone-dependent breast cancer (Perrault, Eisenhauer et al. 1996).
RU486 shows a high level of oral bioavailability (40-70%). After
oral dosing it reaches peak plasma levels after 1-2 hours, with a
plasma half life of 20-30 hours. Concentrations in the blood are
limited by the need for complexing with a specific carrier protein,
with unbound mifepristone being metabolised in the liver.
Metabolism involves demethylation and hydroxylation, with nine
metabolites identified in the rat, four of which have also been
detected in man. The three primary metabolites detected in man are
an N-mono-demethylated compound (RU42633), N-didemethylated
(RU42848) and a hydroxylated compound (RU42698). The fourth is an
N-acetylated derivative of RU42848. The three primary metabolites
show strong progesterone receptor and glucocorticoid receptor
binding activity (Baulieu and Segal 1985).
[0009] RU486 has been administered topically to the eye of rabbits
(Phillips, Green et al. 1984; Tsukahara, Sasaki et al. 1986), in
order to test its ability to lower intraocular pressure due to its
glucocorticoid receptor antagonist activity. Cheeks and Green
(Cheeks and Green 1986) measured the concentration of RU486 in
rabbit ocular tissues after administration of eye drops and showed
that while the vitreous concentration was low, that of the retina
and especially choroid were higher, suggesting that passage of the
drug through the iris root into the suprachoroidal space and beyond
occurs. Therapeutically relevant concentrations of active compound
should reach the choroid and retina after topical administration of
RU486 or related molecule (e.g. a metabolite of RU486) and as such
the preferred embodiment is to have an eye drop formulation.
However, therapeutically effective concentrations may also be
achieved by oral administration (see infra).
[0010] It is possible that special drug delivery systems may be
needed in some cases in order to achieve the required concentration
at the retina and choroid. Examples of technology that might be
employed to this end are sustained release micro or nano particles
such as those used for retinal delivery of budesonide (Kompella,
Bandi et al. 2003), ocular inserts, contact lenses, gel-to-sol
systems, vesicular systems, liposomes, niosomes, mucoadhesive
dosage forms, penetration enhancers, microemulsions, iontophoresis,
dendrimers, all of which are reviewed by Sultana et al. (2006).
SUMMARY OF THE INVENTION
[0011] The present invention is based at least in part on the
realisation that a progesterone antagonist, e.g. a progesterone
receptor modulator, may be used to prevent or treat pathogenic
angiogenesis in the eye, especially VEGF-mediated choroidal
neovascularisation. In this scenario, and without wishing to be
bound by theory, a PR modulator delivered to the eye can reach
sufficiently pharmacologically active PR antagonist concentrations
at the back of the eye to impact on the level of PR activity.
Reduction of the level of activity of the PR then reduces
angiogenesis by modulation of VEGF. Thus, according to the present
invention, a compound having PR antagonist activity is used for
manufacture of a medicament for the treatment of ocular
neovascularisation (in particular Age Related Macular
Degeneration), wherein said compound possesses PR receptor binding
activity that inhibits the agonist activity of progestins.
[0012] According to a first aspect, the invention provides the use
of an agent having progesterone antagonist properties for the
manufacture of a medicament for use in the treatment of a condition
associated with blood vessel formation.
[0013] In preferred embodiments, the invention contemplates the use
of mifepristone (RU486), or a metabolite or pharmaceutically
acceptable derivative thereof, for the treatment of wet AMD. In
such embodiments, the mifepristone (RU486) metabolite may be
selected from RU42698, RU42848 and RU42633. Other preferred
progesterone antagonists are onapristone and asoprisnil (see
infra).
[0014] Also contemplated are pharmaceutical compositions for
topical administration to the eye comprising mifepristone (RU486),
or a metabolite or pharmaceutically acceptable derivative thereof.
In such embodiments, the mifepristone (RU486) metabolite may be
selected from RU42698, RU42848 and RU42633 The mifepristone
(RU486), or metabolite or pharmaceutically acceptable derivative
thereof, is preferably present in an amount sufficient to act as a
PR antagonist on administration of the composition to the eye.
[0015] Also contemplated is an ocular drug delivery system (e.g. a
slow release ocular drug delivery system) comprising mifepristone
(RU486), or a metabolite or pharmaceutically acceptable derivative
thereof. In such embodiments, the mifepristone (RU486), or
metabolite or pharmaceutically acceptable derivative thereof, may
be present in an amount sufficient to act as a PR antagonist on
administration of the composition to the eye.
[0016] Other aspects of the invention are recited in the claims set
out below.
General Preferences and Definitions
[0017] References herein to any particular progesterone antagonist
(and in particular references to particular progesterone receptor
(PR) antagonists, e.g. RU486) include pharmaceutically acceptable
derivatives (e.g. salts) and analogues (e.g. metabolites, such as
the RU486 metabolites RU42633, RU42848 and RU42698) thereof. Thus,
references herein to RU486 (mifepristone) are to be interpreted to
cover RU486 metabolites and pharmaceutically acceptable salts
thereof.
[0018] The term pharmaceutically acceptable derivative as applied
to the progesterone antagonists of the invention define
progesterone antagonists which are obtained (or obtainable) by
chemical derivatization of the parent progesterone antagonists. The
pharmaceutically acceptable derivatives are suitable for
administration to or use in contact with mammalian tissues without
undue toxicity, irritation or allergic response (i.e. commensurate
with a reasonable benefit/risk ratio). Preferred derivatives are
those obtained (or obtainable) by demethylation, hydroxylation,
acetylation, alkylation, glycosidation, esterification or acylation
of the parent progesterone antagonists. The derivatives may be
active per se, or may be inactive until processed in vivo. In the
latter case, the derivatives of the invention act as pro-drugs.
Particularly preferred pro-drugs are ester derivatives which are
esterified at one or more of the free hydroxyls and which are
activated by hydrolysis in vivo. The pharmaceutically acceptable
derivatives of the invention may retain some or all of the activity
of the parent progesterone antagonist. In some cases, the activity
is increased by derivatization. Derivatization may also augment
other biological activities of the progesterone antagonist, for
example bioavailability.
[0019] Moreover, in cases where the progesterone antagonist for use
according to the invention is optically active, the present
invention contemplates all optical isomers, racemic forms and
diastereoisomers of the progesterone antagonist. Thus, references
to any particular progesterone antagonist (e.g. to any particular
PR antagonist) are to be interpreted to encompass racemic mixture
of diastereoisomers, individual diastereoisomers, as a mixture of
enantiomers as well as in the form of individual enantiomers.
[0020] A "pharmaceutical composition" is a solid or liquid
composition in a form, concentration and level of purity suitable
for administration to a patient (e.g. a human or animal patient)
upon which administration it can elicit the desired physiological
changes. Pharmaceutical compositions are typically sterile and/or
non-pyrogenic. The term non-pyrogenic as applied to the
pharmaceutical compositions of the invention defines compositions
which do not elicit undesirable inflammatory responses when
administered to a patient.
DETAILED DESCRIPTION OF THE INVENTION
[0021] The antiprogestin (PR antagonist) activity of a compound for
use in the invention can be determined by the in vitro functional
assay (Edwards et al. (1995); Morgan et al. (2002) J. Med. Chem.
45: 2417-2424) and receptor binding assay (Gill, Lockey et al.
1986; Morgan, Swick et al. 2002). The preferred range of activity
is 0.1 nM to 10 .mu.M (the IC.sub.50 of RU486 by the PR binding
assay method is approximately 6 nM). Other suitable assays are
described in the "Exemplification" section, below.
[0022] Preferred compounds that satisfy the criteria for use in the
invention include RU486 ("mifepristone", Roussel Uclaf, Paris; U.S.
Pat. No. 4,386,085), its monodemethylated and didemethylated
derivatives (RU42633 and RU42848), its alcoholic non-demethylated
derivative (RU42698) and the demethyl and didemethyl derivatives of
RU42698.
[0023] The structures of these compounds are shown below:
##STR00001## ##STR00002##
[0024] Other suitable compounds include onapristone (Schering AG,
Berlin; U.S. Pat. No. 4,780,461), the structure of which is shown
below:
##STR00003##
[0025] Other suitable progesterone antagonists include the steroids
described in the following patents and patent applications: U.S.
Pat. No. 4,609,651, especially the compound lilopristone
(11-beta-(4-dimethylaminophenyl)-17-beta-hydroxy-17-alpha-(3-hydroxy-prop-
-1-(Z)-enzyl-4,9(10)-estradien-3-one); U.S. Pat. No. 5,089,635,
especially the compounds
11-beta-(4-acetylphenyl)-17-beta-hydroxy-17-alpha-(1-propinyl)-4,9-estrad-
ien-3-one and
11-beta-(4-acetylphenyl)-17beta-hydroxy-17-alpha-(3-hydroxy-1(2)-propenyl-
)-4,9-estradien-3-one; U.S. Pat. No. 5,095,129; EP-A 04042831; and
other anti-gestations, e.g., U.S. Pat. No. 4,891,368. Also,
Faslodex ICI 182,780, which has antiprogestin activity (Wu, Liang
et al. 2005), and compounds made by RW Johnson (Palmer, Campen et
al. 2000): tetrahydropyridazines exemplified by RWJ 26819.
Toripristone, ZK 112993, ZK 98299 (onapristone), ZK 98734, ZK
114043, ZK 114863 etc, aglepristone (RU534), RU43044, JNJ 1250132
(Allan, Palmer et al. 2006), lilopristone, SPRMs exemplified by
asoprisnil (Schubert, Elger et al. 2005), ZK 230211 (Fuhrmann,
Hess-Stumpp et al. 2000), RTI 3021-012 and RTI 3021-022 (Wagner,
Pollio et al. 1999), CDB-2914 (Hild, Reel et al. 2000), CDB-4124
and CDB-4453 (Attardi, Burgenson et al. 2002), 16 alpha substituted
RU486 (Wagner, Pollio et al. 1996) and compounds in papers
(Giannoukos, Szapary et al. 2001; Leonhardt and Edwards 2002;
Sathya, Jansen et al. 2002; Chabbert-Buffet, Meduri et al. 2005).
Also included are 17-spirofuran-3'-ylidene steroids (U.S. Pat. No.
5,292,878) 11-arylsteroid compounds as disclosed in U.S. Pat. No.
4,921,845, dihydroquinolone (U.S. Pat. No. 5,688,810),
cyclocarbamate derivatives as disclosed in U.S. Pat. No. 6,509,334,
the PR ligands disclosed in the U.S. Pat. No. 5,693,646, U.S. Pat.
No. 5,696,127, U.S. Pat. No. 5,994,544, U.S. Pat. No. 6,358,947,
U.S. Pat. No. 6,713,478, U.S. Pat. No. 6,566,372, U.S. Pat. No.
6,436,929, U.S. Pat. No. 6,358,948, U.S. Pat. No. 7,081,457, the
5-(1',1'-cycloalkyl/alkenyl)methylidene
1,2-dihydro-5H-chromeno[3,4-f]quinolines and 5-cycloalkenyl
5H-chromeno[3,4-f]quinolines as disclosed by Zhi, et al (U.S. Pat.
Nos. 7,084,151 and 7,071,205). Also included are the selective
progesterone receptor modulators disclosed in U.S. Pat. Nos.
7,084,151 and 7,071,205 and the PR antagonists described by Jones,
et al., (Edwards, Zhi et al. 1998; Hamann, Winn et al. 1998;
Tegley, Zhi et al. 1998; Zhi, Tegley et al. 1998; Zhi, Tegley et
al. 1998; Zhi, Tegley et al. 1999; Zhi, Tegley et al. 2000; Zhi,
Ringgenberg et al. 2003; Zhi, Tegley et al. 2003; Zhi, Tegley et
al. 2003), the amide PR ligand described by Combs, et al., (Combs,
Reese et al. 1995; Combs, Reese et al. 1995), the PR antagonists
described by Hamann, et al., (Hamann, Farmer et al. 1995; Hamann,
Farmer et al. 1996; Jones, Pathirana et al. 1996; Hamann, Winn et
al. 1998) and the tatrahydronaphthofuranones and
tetrahydrobenzindolones described by Kurihara, et al., (Kurihara,
Shinei et al. 2006; Shinei, Kurihara et al. 2006).
[0026] Thus, included are the following compounds:
TABLE-US-00001 Compound Structure Wyeth 6 ##STR00004## Wyeth 4j
##STR00005## Wyeth 4g ##STR00006## Wyeth 4h ##STR00007## LG120753
##STR00008## Asoprisnil ##STR00009## LG121046 ##STR00010##
Onapristone ##STR00011##
[0027] Other suitable compounds include those listed below:
##STR00012## ##STR00013##
[0028] Other suitable compounds are described in WO99/25360,
EP277676, EP289073, EP321010, EP549041 and EP582338 (the disclosure
of which relating to progesterone antagonists is hereby
incorporated herein by reference).
[0029] The invention can be used for the treatment of
neovascularisation in age-related macular degeneration (AMD),
including in particular wet AMD, ocular histoplasmosis syndrome,
pathologic myopia, angioid streaks, idiopathic disorders,
choroiditis, choroidal rupture, overlying choroid nevi, Best's
disease, Stargardt's disease, Vogt-Koyanagi-Harada syndrome,
toxoplasmosis, certain inflammatory diseases, sickle cell disease,
diabetic retinopathy and other proliferative retinopathy,
retinopathy of prematurity, neovascular glaucoma, sarcoidosis,
syphilis, pseudoxanthoma elasticum, vein or artery occlusion,
carotid obstructive disease, chronic uveitis/vitritis,
mycobacterial infection, Lyme's disease, Eale's disease, systemic
lupus erythomatosus, Behcet's disease, infections causing
retinitis, optic pits, par planitis, chronic retinal detachment,
hyperviscosity syndromes, capillary haemangioma including von
Hippel-Lindau disease, trauma and post-laser complication.
[0030] An enhanced effect on retinal/choroidal/corneal NV and/or
age-related macular degeneration may be achieved by
co-administering a PR antagonist with another agent that is used
for these conditions, such as macugen, lucentis, VEGF inhibitors,
VEGF receptor tyrosine kinase inhibitors, protein kinase C
inhibitors, inhibitors of other angiogenic proteins such as
insulin-like growth factor and angiopoietin, recombinant
angiostatic factors such as pigment epithelium-derived factor
(PEDF), thrombospondin, and endostatin, somatostatin analogues,
corticosteroids (such as but not limited to triamcinolone and
anecortave acetate), statins (inhibitors of HMG-CoA reductase),
squalamine lactate, thiamine and its analogues, angiotensin
receptor blockers and rapamycin. The PR antagonist may also be
administered as an adjunct to laser coagulation, photodynamic
therapy (including visudyne), or cryotherapy.
Formulation and Posology
[0031] In general, the active compound may be administered by known
means, in any suitable formulation, by any suitable route. For oral
administration, it is preferably formulated as a tablet, troche,
lozenge, capsule, emulsion, syrup or elixir. A compound of this
invention is preferably administered to the eye topically or by an
ocular drug delivery system.
[0032] Compositions for oral administration include known
pharmaceutical forms for such administration, for example lozenges,
pastilles, dispersible tablets, powders or granules or as a liquid
for spraying into the mouth. Compositions intended for oral use may
be prepared according to any method known to the art for the
manufacture of pharmaceutical compositions, and such compositions
may contain one or more agents selected from the group consisting
of sweetening agents, flavouring agents, colouring agents and
preserving agents in order to provide pharmaceutically elegant and
palatable preparations. Tablets contain the active ingredient in
admixture with non-toxic pharmaceutically acceptable excipients
which are suitable for the manufacture of tablets. These excipients
may be, for example, inert diluents, such as calcium carbonate,
sodium carbonate, lactose, calcium phosphate or sodium phosphate;
granulating and disintegrating agents, for example corn starch or
alginic acid; binding agents, for example starch gelatin, acacia,
microcrystalline cellulose or polyvinyl pyrrolidone; and
lubricating agents, for example magnesium stearate, stearic acid or
talc. The tablets may be uncoated or they may be coated by known
techniques to delay disintegration and absorption in the
gastrointestinal tract and thereby provide a sustained action over
a longer period. For example, a time-delay material such as
glyceryl monostearate or glyceryl distearate may be employed.
[0033] For oral administration, the composition may be in any form
that will release the active agent, when held in the mouth, whether
for a short time or for a matter of hours. It may be malleable and
non-disintegrating, and/or chewable or dispersible. Preferred
examples of such compositions are gums, as well as wafers and
dispersible tablets (described above). A flavorant will typically
be included. It is particularly desirable if the flavorant has
mucolytic properties. An example of such a flavorant is
menthol.
[0034] Aqueous suspensions contain the active materials in
admixture with excipients suitable for the manufacture of aqueous
suspensions. Such excipients are suspending agents, for example
sodium carboxymethylcellulose, methylcellulose,
hydroxypropylmethylcellulose, sodium alginate, polyvinyl
pyrrolidone, gum tragacanth and gum acacia; dispersing or wetting
agents may be a naturally occurring phosphatide, for example
lecithin, or condensation products of an alkylene oxide with fatty
acids, for example polyoxyethylene stearate, or condensation
products of ethylene oxide with long-chain aliphatic alcohols, for
example heptadecaethyleneoxycetanol, or condensation products of
ethylene oxide with partial esters derived from fatty acids, for
example polyoxyethylene sorbitan monooleate. The aqueous
suspensions may also contain one or more preservatives, for example
ethyl or n-propyl p-hydroxybenzoate, one or more colouring agents,
one or more flavouring agents, and one or more sweetening agents,
such as sucrose or saccharin.
[0035] Oily suspensions may be formulated by suspending the active
ingredient in a vegetable oil, for example arachis oil, olive oil,
sesame oil or coconut oil, polyoxyethylene hydrogenated castor oil,
fatty acids such as oleic acid, or in a mineral oil such as liquid
paraffin or in other surfactants or detergents. The oily
suspensions may contain a thickening agent, for example beeswax,
hard paraffin or cetyl alcohol. Sweetening agents, such as those
set forth above, and flavouring agents may be added to provide a
palatable oral preparation. These compositions may be preserved by
the addition of an antioxidant such as ascorbic acid.
[0036] Dispersible powders and granules suitable for preparation of
an aqueous suspension by the addition of water provide the active
ingredient in admixture with a dispersing or wetting agent,
suspending agent and one or more preservatives. Suitable
sweetening, flavouring and colouring agents may also be
present.
[0037] Compositions for use in the invention may be formulated in a
manner known to those skilled in the art so as to give a controlled
release, for example rapid release or sustained release, of the
compounds of the present invention. Pharmaceutically acceptable
carriers suitable for use in such compositions are well known in
the art. The compositions of the invention may contain 0.1-99% by
weight of active compound. The compositions of the invention are
generally prepared in unit dosage form. Preferably, a unit dose
comprises the active ingredient in an amount of 0.001 to 100 mg.
The excipients used in the preparation of these compositions are
the excipients known in the art.
[0038] Appropriate dosage levels may be determined by any suitable
method known to one skilled in the art. It will be understood,
however, that the specific dose level for any particular patient
will depend upon a variety of factors including the activity of the
specific compound employed, the age, body weight, general health,
sex, diet, time of administration, route of administration, rate of
excretion, drug combination and the severity of the condition to be
treated. Preferably, the active agent is administered at a
frequency of 1 to 4 times per day.
[0039] The compositions may be formulated in a manner known to
those skilled in the art so as to give adequate delivery to the
back of the eye, which may be by regular dosing, such as with an
eye drop, or may using a delivery system to give a controlled
release, such as slow release, of the compounds in the present
invention. Pharmaceutically acceptable carriers suitable for use in
such compositions are well known in the art. The compositions of
the inventions may contain 0.001-99% by weight of the active
compound. The compositions of the invention are generally prepared
in unit dosage form. Preferably, a unit dose comprises the active
ingredient in an amount of 0.001 to 500 mg. The excipients used in
preparation of these compositions are the excipients known in the
art.
[0040] Appropriate dosage levels may be determined by any suitable
method known to one skilled in the art. It will be understood,
however, that the specific dosage level for any particular patient
will depend on a variety of factors including age, body weight,
general health, sex, diet, time of administration, route of
administration, rate of excretion, drug combination and the
severity of the complaint. Preferably the active compound is
administered at a frequency of 1 to 4 times per day for topical
administration, or less often if a drug delivery system is used. A
typical daily dosage for topical treatment is a formulation
containing 0.001-10% active ingredient.
[0041] The pharmaceutical compositions of the invention may be
administered by any means known to those skilled in the art for
treatment of eye diseases. They are preferably administered in the
form of aqueous solutions, suspensions, or gels of the active
compound in the form of drops of liquid, liquid washes, sprays or
ointments, or gel. Alternatively the active compounds may be
applied to the eye via liposomes or other ocular delivery system.
The compositions contain the active materials in admixture with
excipients suitable for the manufacture of aqueous solutions,
suspensions and gels.
[0042] Components of the composition may be chosen from any of
those used in or capable of being used in a pharmaceutical
formulation, especially those designed for topical administration
to the eye. A non-exclusive list of components includes
preservatives, stabilizers, chelating agents, dyes, antibiotics,
antimicrobials, and anti-fungal agents. Preservatives including,
but not limited to, benzalkonium chloride, thimerosal, and phenyl
mercuric nitrate may be used in a range between about 0.001 to 1
percent by weight. The compositions of the present invention may
further comprise pharmaceutically acceptable carriers, excipients,
gels, solutions, or diluents suitable for topical ophthalmic
administration, and may include pharmaceutically acceptable
polymeric suspension agents, solubilising agents, solvents and
surfactants. Suitable carriers or excipients include, but are not
limited to, calcium carbonate, calcium phosphate, various sugars
(e.g. mannitol), starches, cellulose derivatives, gelatin,
N-lauroylsarcosine, non-ionic surfactants, such as polysorbates,
alcohols, such as propylene glycol and glycerol, and water soluble
polymers such as polyethylene glycol. Polymeric suspension agents
may be used, comprising one or more polymers, including dextrans,
polyethylene glycols, polyvinylpyrrolidone, polysaccharide gels,
Gelrite..RTM., tyloxapol, octoxynols, cellulosic polymers like
hydroxypropyl methylcellulose (hypromellose), and
carboxyl-containing polymers such as acrylic acid-containing
polymers and vinyl-containing polymers (e.g. polyvinylpyrollidone).
Particularly preferred polymers include polycarbophil, the
DuraSite..RTM.. polymeric delivery system (InSite Vision, Inc.,
Alameda, Calif.), and mucomimetic polymers (see, e.g., U.S. Pat.
No. 5,932,572). Dispersing or wetting agents may be a naturally
occurring phosphatide for example lecithin, or condensation
products of an alkylene oxide with fatty acids, for example
polyoxyethylene stearate, or condensation products of ethylene
oxide with long chain aliphatic alcohols, for examples
heptadecaethyleneoxycetanol or polyoxyethylene hydrogenated castor
oil, or condensation products of ethylene oxide with partial esters
derived from fatty acids, for example polyoxyethylene sorbitan
monooleate. Other components may include a chelating agent such as
disodium edetate, and complexing agents such as
hydroxypropyl-beta-cyclodextrin. Antioxidants may be added to
protect the active component from oxidation during storage.
Examples of such antioxidants include vitamin E and analogues
thereof, ascorbic acid, and butylated hydroxytoluene (BHT). The pH
of the inventive compositions is preferably between about 6 and
about 8, and may be adjusted for the particular compound(s) used.
Purified water USP and various acids and bases suitable for
ophthalmic use, or combinations of acids and bases, may be used for
adjusting the pH of the compositions. Non-limiting examples of
acids and bases include acetic acid, boric acid, citric acid,
lactic acid, phosphoric acid, hydrochloric acid, sodium hydroxide,
sodium phosphate, sodium borate, sodium citrate, sodium acetate,
sodium lactate, and TRIS.
[0043] The osmotic pressure of the compositions may be adjusted by
methods known in the art to be between about 40 to about 400
milliosmolar (mOsM), more preferably between about 100 to about 300
mOsM. A preferred method of adjusting osmotic pressure is the
addition of physiologically and ophthalmically acceptable salts.
Sodium chloride, which approximates physiological fluid, is the
preferred salt, for use in concentrations ranging from about 0.01
to about 1 percent by weight, or any value in that range.
Preferably, the concentration is between about 0.1 to about 1
percent. Equivalent amounts of one or more salts made up of cations
such as potassium, ammonium and the like and anions such as
chloride, citrate, ascorbate, borate, phosphate, bicarbonate,
sulfate, thiosulfate, bisulfite and the like, e.g., potassium
chloride, sodium thiosulfate, sodium bisulfite, ammonium sulfate,
and the like, can also be used in addition to or instead of sodium
chloride to achieve osmotic pressures within the above-stated
ranges. An example of a preferred composition is for an ocular
solution containing the compound in aqueous solution with
hydroxypropyl-beta-cyclodextrin, polyethylene glycol 400, and
hydroxypropylmethylcellulose.
[0044] Examples of treatment scenarios envisaged for PR antagonism
are as follows: (a) topical administration to the eye of RU486, or
RU486 metabolite (RU42698, RU42848 and RU42633) or selective PR
antagonist to individuals with CNV; (b) administration using an
ocular drug delivery system of RU486, or RU486 metabolite (RU42698,
RU42848 and RU42633) or selective PR antagonist to individuals with
CNV; (c) topical administration to the eye of RU486, or RU486
metabolite (RU42698, RU42848 and RU42633) or selective PR
antagonist to individuals with drusen characteristic of early
age-related macular degeneration to reduce the risk of developing
CNV; (d) administration using an ocular drug delivery system of
RU486, or RU486 metabolite (RU42698, RU42848 and RU42633) or
selective PR antagonist to individuals with drusen characteristic
of early age-related macular degeneration to reduce the risk of
developing CNV; (e) topical administration to the eye of RU486, or
RU486 metabolite (RU42698, RU42848 and RU42633) or selective PR
antagonist to individuals with retinal neovascularization; (f)
administration using an ocular drug delivery system of RU486, or
RU486 metabolite (RU42698, RU42848 and RU42633) or selective PR
antagonist to individuals with retinal neovascularization; (g)
topical administration to the eye of RU486, or RU486 metabolite
(RU42698, RU42848 and RU42633) or selective PR antagonist to
individuals with corneal neovascularization; (h) administration
using an ocular drug delivery system of RU486, or RU486 metabolite
(RU42698, RU42848 and RU42633) or selective PR antagonist to
individuals with corneal neovascularization; (i) topical
administration to the eye of any of the above compounds possessing
PR antagonist activity given in combination with other agents that
reduce the activity of VEGF (e.g., VEGF inhibitor, VEGF receptor
tyrosine kinase inhibitors), protein kinase C inhibitors,
inhibitors of other angiogenic proteins such as insulin-like growth
factor and angiopoietin, recombinant angiostatic factors such as
pigment epithelium-derived factor (PEDF), thrombospondin,
endostatin, corticosteroids (such as but not limited to
triamcinolone and anecortave) and statins (inhibitors of HMG-CoA
reductase). These other agents may be given topically to the eye or
introduced directly into the eye via injections or implant devices,
or systemically.
Exemplification
[0045] The invention will now be described with reference to
specific studies. These are merely exemplary and for illustrative
purposes only: they are not intended to be limiting in any way to
the scope of the monopoly claimed or to the invention described.
The studies provide evidence of utility of the present
invention.
Zebrafish Angiogenesis Assays
[0046] Zebrafish (Danio rerio) are ideal organisms for the
investigation of blood vessel growth because they are transparent
in the embryonic and larval stages of development so that blood
vessels marked with a fluorescent marker can clearly be seen in
vivo in the live animal. Zebrafish blood vessel development is well
characterised and vessels can be visualised by microangiography or
by expressing green fluorescent protein in blood vessels (Isogai et
al. (2001) Dev Biol 230(2): 278-301; Covassin et al. (2006) Proc
Natl Acad Sci USA 103(17): 6554-9). Zebrafish are also ideal
vertebrate organisms in which to screen for active small molecules
because the larvae can be treated with compound simply by immersion
of the fish in compound solution in wells of a standard multi-well
plate (12, 24 or 96-wells).
[0047] Zebrafish protein sequences bear remarkable similarity to
the cognate human protein sequences, especially across important
functional domains (Goldsmith (2004) Curr Opin Pharmacol 4(5):
504-12; Peterson at al. (2004) Nat Biotechnol 22(5): 595-9). In
particular, zebrafish contain a progesterone receptor gene (NCBI
Accession Number AAY85275.1) which is 67% identical and 96% similar
to the human progesterone receptor over the ligand binding domain.
Moreover, the VEGF signalling pathway is highly conserved between
zebrafish and mammals (Covassin et al. (2006) Proc Natl Acad Sci
USA 103(17): 6554-9). Thus, compounds that inhibit zebrafish blood
vessel formation are likely to be anti-angiogenic in man.
[0048] Expression of the zebrafish PR can be detected as early as
48 hours post fertilisation and an assay was therefore developed to
investigate the effects of compounds on blood vessel formation in
zebrafish after this time. Previous studies on inhibition of blood
vessel growth have focussed on formation of intersegmental vessels
(Serbedzija et al. (1999) Angiogenesis 3(4): 353-9; Parng at al.
(2002) Assay Drug Dev Technol 1(1 Pt 1): 41-8) but these particular
vessels are specified and begin to form prior to the time when the
PR is expressed in the zebrafish embryo. Accordingly,
later-developing vessels around the gut, the vascular plexus of the
mid gut which branches from the subintestinal vein and
supraintestinal artery, were monitored.
Methods
[0049] Methods have been developed for visualisation of zebrafish
blood vessels by expression of green fluorescent protein in the
vessels of transgenic fish. The lines of transgenic fish most
frequently used for this are those expressing green fluorescent
protein (GFP) under control of the zebrafish flit promoter (Lawson
and Weinstein (2002) Dev Biol 248(2): 307-18) or flk1 promoter (Jin
et al. (2005) Development 132(23): 5199-209).
[0050] A similar method was developed for screening of compounds
using a line of fish that express GFP under control of an enhancer
that drives expression in blood vessels, spinal cord and eyes. The
fish are a cross between the et19.1 enhancer:Gal4 line (Scott et
al. (2007) Nat Methods 4(4): 323-6) and a UAS:GFP line made
in-house in a TL zebrafish background. Transgenic zebrafish embryos
or larvae expressing GFP in the blood vessel walls were treated
with test compounds by immersion in aqueous medium containing the
compound at the required concentration, for a period of one or more
days. At the end of the incubation period the fish were immobilised
on their sides and the organisation and structure of the
vasculature was visualised by fluorescent microscopy, which is
possible because of the transparency of zebrafish larvae. The
vessels can be identified by comparison with the online Interactive
Atlas of Zebrafish Vascular Anatomy (Isogai et al. (2001) Dev Biol
230(2): 278-301).
[0051] Vessels such as the dorsal aorta, posterior cardinal vein,
caudal artery, caudal vein, intersegmental vessels, dorsal
longitudinal anastomotic vessels and the gut vessel plexus were
assessed in each fish and compared to the control group. The gut
vessel plexus formed during the time at which progesterone receptor
is expressed and these were the vessels that were shown to be
inhibited by progesterone receptor antagonists. Development of
these vessels was shown to be VEGF dependent because vessel
formation was inhibited by incubation with VEGF receptor inhibitor
SU5416. A subjective score was given to the fore/mid gut vessel
plexus (by assessment of the number of visible vessels and extent
of spread of the plexus), ranging from "3" for a normal extensively
vascularised gut, to "0" for a complete absence of any branching of
vessels around the gut. The intestinal vessel plexus forms between
3 and 5 days post fertilisation (d.p.f.) and so these assays were
usually scored at 5 d.p.f. The vessels could also be quantified by
digital photography and quantification of fluorescence levels or
number of vessels in the region of interest.
[0052] Those skilled in the art will appreciate that other methods
for visualisation of blood vessels may be used, including: (1)
fluorescent angiography (injection of a fluorescent marker molecule
such as FITC-dextran into the vascular system in live fish and
visualisation of the vessels by fluorescent microscopy), using
methods such as those described by (Weinstein et al. (1995) Nat Med
1(11): 1143-7; Peterson et al., (2004) Nat Biotechnol 22(5):
595-9); (2) staining of vessels with antibody specific for blood
vessels (such as anti-Tie-2 or anti-VEGF receptor antibody); and
(3) staining for endogenous alkaline phosphatase activity (Habeck
et al., (2002) Curr Biol 12(16): 1405-12; Serbedzija et al. (1999)
Angiogenesis 3(4): 353-9; Parng et al. (2002) Assay Drug Dev
Technol 1(1 Pt 1): 41-8). It is also to be understood that an
alternative to assessing developmental angiogenesis (occurring in
embryos/larvae) is the assessment of angiogenesis in adult fish,
such as during regeneration of the tail fin. Suitable methods for
use in such assays are described for example by Bayliss et al.
(2006) Nat Chem Biol 2(5): 265-73). Angiogenesis can be scored by
using gene expression as a surrogate, for example by quantification
of mRNA levels for genes involved in angiogenesis (e.g. VEGF and
its receptors). Suitable methods for use in such assays include
RT-PCR or RNA in situ hybridization or quantification of protein
(using methods such as Western blotting or ELISA).
[0053] It will also be appreciated that compounds may also be
tested in cell culture assays in vitro using mammalian endothelial
cells, in particular a tubule development assay in normal human
endothelial cells co-cultured with interstitial cells, as disclosed
in U.S. Pat. No. 6,225,118, GB2331763 and EP1023599. These cultures
spontaneously develop a network of capillary-like tubules after
10-14 days in culture. Tubule development is believed to closely
mimic in vivo angiogenesis and is enhanced by known stimulators of
angiogenesis and suppressed by known angiogenesis inhibitors, both
in a dose-dependent manner (Bishop et al., (1999) Angiogenesis
3(4): 335-44; Donovan et al. (2001) Angiogenesis 4(2): 113-21;
Clarke et al. (2006) Cancer Res 66(7): 3504-12). Other suitable in
vitro assays include assessment of proliferation, migration,
invasion and wound healing in endothelial cell cultures, and a rat
aortic ring culture assay.
Mammalian Angiogenesis Assays
[0054] Those skilled in the art will also appreciate that compounds
for use according to the invention can also be identified using
mammalian angiogenesis assays. In particular, compounds suitable
for use in the treatment of wet AMD can be identified using assays
based on inhibition of laser-induced choroidal neovascularisation
in mammalian models (such as the laser-induced CNV model). This
involves laser photocoagulation of the RPE and choroid, resulting
in a wound response that includes formation of a neovascular
lesion. This is usually performed in rodents or primates. The
protocol used is essentially as described by Kinose et al. (2005)
Mol Vis 11: 366-73). Methods for assessment of neovascular lesions
include angiography with fluorescein or indocyanine green, detected
as regions of leakage from vessels by fundus photography using an
instrument such as a confocal scanning laser opthalmoscope (see
e.g. Takehana et al. (1999) Invest Opthalmol Vis Sci 40(2): 459-66;
Koh et al., (2004) Invest Opthalmol Vis Sci 45(2): 635-40). Also,
neovessels can be detected by ex vivo analysis of flatmounts after
infusion with FITC dextran or similar agent (Edelman and Castro
(2000) Exp Eye Res 71(5): 523-33), or by staining with a specific
antibody, histochemical visualisation (Semkova et al. (2003) Invest
Opthalmol Vis Sci 44(12): 5349-54) or by histological analysis of
stained sections of retinal tissue (Takehana et al., (1999) Invest
Opthalmol Vis Sci 40(2): 459-66; Yanagi et al. (2003) Invest
Opthalmol Vis Sci 44(2): 751-4; Koh et al. (2004) Invest Opthalmol
Vis Sci 45(2): 635-40). Other suitable in vivo angiogenesis assays
for measurement of inhibition of angiogenesis at other sites
include retinal and corneal angiogenesis models in rodents and
rabbits and mammalian models that involve insertion into various
regions of the body of an implant containing angiogenic factors
such as VEGF or basement membrane extracts.
Results
[0055] Assays using Tg 043/050 uas:GFP+/-enhancer:gal4+/-.times.TL
("panretinal" enhancer) fish, incubated with various compounds (see
Table below) from 24 h.p.f. to 5 d.p.f. (unless otherwise stated),
then scoring the extent of the gut vessel plexus by microscopy. A
score of 3 represents a good, fully formed gut vessel plexus, with
20 or more branched vessels; 2 represents as slightly less well
formed plexus with 10-19 vessels; 1 represents a poorly formed
plexus with 1-9 vessels; 0 represents a complete absence of visible
gut vessels. Each of the compounds was tested in a 24 hour MTC
assay in the TL strain of wild type fish (24-48 h.p.f.). The MTC
from this assay was used as the highest concentration for assays in
the transgenic fish, with half-log intervals.
[0056] RU486 inhibition was dose-dependent (data not shown) and was
rescued by progesterone (10 .mu.M of each), suggesting that the
effects of RU486 in this system are regulated by progesterone
receptor. RU42698 also showed concentration-dependent inhibition of
vessels at 30-100 .mu.M. RU42633 concentration response curves (1-5
dpf) showed clear concentration-dependent inhibition of gut vessels
with best inhibition at 30 .mu.M. Wyeth 4h also showed
concentration-dependent inhibition, maximal at 30 .mu.M (data not
shown). LG120753 showed concentration-dependent inhibition at 0.3-3
.mu.M.
TABLE-US-00002 concs Effective MTC No. of scored Inhibition of gut
concs Compound (.mu.M) assays (.mu.M) vessel formation (.mu.M)
RU486 10-30 3 0.3-10 +++ 3-10 RU42633 30 2 0.3-30 +++ 3-10 RU42698
100 3 1-100 ++ 30-100 RU42848 100 2 1-100 + (10-30) Wyeth 6 100 3
1-100 + (10), 30 Wyeth 4j 1-3 3 0.1-3 + 1-3 Wyeth 4g 3 2 0.1-3 ++
0.3, 3 Wyeth 4h 30 2 0.3-30 ++ 1-30 LG120753 3 2 0.03-3 + 1-3
Asoprisnil 10 2 0.3-30 + 10 LG121046 3-10 2 0.1-10 ++ 1-10 Key to
extent of inhibition: +++ = inhibition of vessels equivalent to
that of 10 .mu.M RU486 (which has a mean score of 1 to 1.5). ++ =
inhibition of vessels but to a lesser extent that RU486. + =
slightly fewer vessels than in controls but much less inhibition
than with RU486. - = no detectable inhibition.
EQUIVALENTS
[0057] The foregoing description details presently preferred
embodiments of the present invention. Numerous modifications and
variations in practice thereof are expected to occur to those
skilled in the art upon consideration of these descriptions. Those
modifications and variations are intended to be encompassed within
the claims appended hereto.
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